A high affinity, low capacity (specific) binder of triiodothyronine (T3) has previosly been obtained from mitochondrial membranes of rat as well as human liver and kidney. Fractionation of mitochondrial membrane protein entails an initial step of Sephadex G-200 gel filtration. The large molecular weight fraction (approximately 150,000) binds T3 with association constant (KA) of 10 to the 11th power M to the minus 1 power by Scatchard plot on addition of graded increments of T3 in the picogram range. The displacement of tracer (125I)-T3 from cell nuclei after injection in vivo of nonradioactive T3 has been observed repeatedly. In contrast, displacement of tracer T3 from intact mitochondria has not previously been reported, leading to the supposition that saturable receptors exist only in the cell nucleus. The present work shows for the first time that in vivo displacement of tracer (125I)-T3 is regularly observed when loading doses of nonradioactive T3 are injected into the tail vein of the rat, provided the mitochondrial receptor is partially purified by gel filtration. Failure of displacement from intact mitochondria is attributed to abundant nonspecific sites of the outer mitochondrial membrane. Current evidence suggest that the mitochondrial receptor for T3 arises from the inner membrane, the site of oxidative phosphorylation. Purification of the binding macromolecule by affinity chromatography yields a lipoprotein containing lecithin, phosphatidyl ethanolamine, cardiolipin, and other constituents of mitochondrial membrane. KA remains 10 to the 11th power M to the minus 1 power throughout purification. Organ distribution suggests a physiological role; no saturable receptors have been found in mitochondria of adult brain, spleen or testis. In light of other evidence on rapid stimulation of ATP formation in hepatic mitochondria of hypothyroid rats within 30 minutes after intravenous injection of nanogram doses of T3, it is inferred that the hormone stimulates oxidative phosphorylation directly by an extranuclear pathway.